1. Field of the Invention
This invention relates generally to analytical tests for the determination of peroxidatively active substances in test samples, and particularly to an improved test composition and device for such determinations having enhanced storage stability, as well as to a method for making and using the improved composition and device.
Many analytical methods are available for detecting the presence of peroxidatively active substances in biological samples such as urine, fecal suspensions and gastrointestinal contents. Hemoglobin and its derivatives are typical examples of such "peroxidatively active" substances because they behave in a manner similar to the enzyme peroxidase. Thus, such substances have also been referred to as pseudoperoxidases, i.e., enzyme-like in that they catalyze the redox reaction between peroxides and such indicator compounds as benzidine, o-tolidine, 3,3',5,5'-tetramethylbenzidine, 2,7-diaminofluorene or similar substances, thereby producing a detectable response such as color change. For example, most diagnostic assays for determining the presence of occult blood in urine rely on this pseudoperoxidase activity.
2. Background Art
The analytical test methods which, over the years, have relied on enzyme-like catalysis of the peroxidative oxidation of colorforming indicators, include wet chemical or solution procedures as well as the so-called "dip-and-read" type, reagent-bearing strip devices. Of the former, a typical example is set forth in Richard M. Henry, et al., Clinical Chemistry Principles and Techniques, 2nd ed., (Hagerstown, Maryland: Harper and Row, 1974), pp. 1124-1125. This procedure involves the use of glacial acetic acid (buffer), diphenylamine (indicator) and hydrogen peroxide. While such wet methods have proven analytical utility, they nevertheless have shortcomings, such as poor reagent stability and inadequate sensitivity.
Another method for the determination of peroxidatively active substances, and one presently preferred by most clinical assayists, employs the "dip-and-read", solid phase reagent strip device. Typical of such devices are those commercially available from the Ames Division of Miles Laboratories, Inc. under the trademark HEMASTIX.RTM.. They comprise, in essence, a porous paper matrix affixed to a plastic strip or handle. The matrix is impregnated with a buffered mixture of an organic hydroperoxide, for example, cumene hydroperoxide, and an indicator compound. Upon immersion in a liquid containing a peroxidatively active analyte such as hemoglobin, myoglobin, erythrocytes or other pseudoperoxidases, a blue color develops in the matrix, the intensity of which is proportional to the concentration of the analyte in the sample. By comparing the color developed in the matrix to a standard color chart, the assayist can determine, on a semiquantitative basis, the amount of the analyte which is present.
Reagent strips possess a number of advantages over wet chemistry methods, for example, greater ease of use because neither the preparation of reagents nor attendant apparatus is required, and greater comparative stability of reagents because of the dry, solid reagent state, resulting in improved accuracy, sensitivity and economy. However, a serious disadvantage of many conventional, presently-available reagent strip test devices is limited "shelf-life", i.e., lack of storage stability over prolonged periods following manufacture, resulting in markedly decreased reactivity to the presence of peroxidatively active analytes when the devices are eventually used. Thus, because analytical tools such as reagent strips usually are not used immediately after manufacture, but stored for varying periods of time before use, and because too long a period between manufacture and use of conventional reagent strips can result in severe losses of reactivity and concomitant false negative test results, enhanced shelf-life can be a marked asset: the better the shelf-life, the more reliable will be the analytical results obtained.
Conventional solid phase reagent strip devices for determining peroxidatively active substances, e.g., occult blood or hemoglobin in urine, typically utilize as an indicator system the porphyrin-catalyzed oxidation of a benzidine-type indicator, for example, o-tolidine or 3,3',5,5'-tetramethylbenzidine, by an organic hydroperoxide, such as cumene hydroperoxide. Such conventional test strips, however, are known to be particularly prone to loss of reactivity during prolonged storage, or storage at elevated temperatures--a phenomenon which is believed to be due either to volatility or chemical degradation of one or more reagent ingredients of the strip. Not only have substantial losses of reactivity been observed in such conventional reagent strips following storage at ambient temperatures, but those losses appear to be greatly accentuated, and the rate of loss accelerated, by storage at elevated temperatures. Possible explanations for the losses of reactivity in reagent strips are: (1) key ingredient(s) of the reagent composition decompose or volatilize, so that the level of ingredient(s) falls below the minimum level necessary to maintain adequate reactivity; and (2) two or more ingredients in the strip interact deleteriously, producing one or more new species which are unreactive or inhibitory.
Attempts to stabilize the reactivity of reagent compositions, and solid phase strip devices made therefrom for determining peroxidatively active substances, have followed various lines of approach. For example, U.S. Pat. No. 3,092,463 to Adams, Jr. et al., discloses an improved test composition and device for detecting occult blood in body fluids. The composition comprises an organic hydroperoxide encapsulated or entrapped in microspherical bubbles of a colloid substance, an indicator or dye precursor capable of accepting transfer of oxygen from the organic hydroperoxide to produce a color response induced by the catalytic action of the prosthetic group of hemoglobin, and a buffer for maintaining the pH of the substance being tested within the range of from 4 to 6.5. This patent discloses that the colloid substance, for example, polyvinyl alcohol, gelatin, gum arabic or carboxy vinyl polymer, can provide stabilization of the reactivity of preferred embodiments of the test device produced from the composition even after 300 hours storage at 75.degree. C., whereas similar devices prepared without encapsulation of the hydroperoxide lost considerable reactivity after 24 hours at 50.degree. C.
Other disclosures of stabilized test compositions and devices include the approach of U.S. Pat. No. 3,252,762 to Adams, Jr. et al., wherein the organic hydroperoxide is encapsulated in a colloidal material such as gelatin which is hardened by fixing with a dialdehyde polysaccharide. Such compositions, containing a hydroperoxide so encapsulated, a suitable indicator and a buffer, are said to exhibit enhanced stability under various adverse temperature conditions.
Still further disclosed attempts at stabilization of reagent strip devices include a recitation in Chemical Abstracts, Vol. 85, p. 186 (1976), describing a two-dip method for preparing reagent strips containing o-tolidine and phenylisopropyl hydroperoxide. This disclosure reports preparation of a solution of the indicator (o-tolidine.2HCl) and polyvinyl pyrrolidone in ethanol. To this solution is added a small amount of surfactant and enough citrate buffer to provide a pH of 3.7, whereafter filter paper strips impregnated with ethyl cellulose are dipped in the solution and dried. The impregnated filter paper is subsequently dipped into a second solution containing 1,4-diazabicyclo[2,2,2]octane, phenylisopropyl hydroperoxide and polyvinyl pyrrolidone, dissolved in an ethanol-toluene mixture. The thrust of this work appears directed toward stabilization of the peroxide and indicator combination through the use of the bicyclooctane derivative and the polyvinylpyrrolidone.
A similar method is disclosed in U.S. Pat. No. 3,853,471. This patent discloses the use of phosphoric or phosphonic acid amides where the substituent amido groups are primarily N-morpholine radicals.
Other approaches to stabilized reagent compositions include those of U.S. Pat. No. 4,071,317, wherein various diluent compounds, such as a mixture of dimethyl sulfone and N,N-dimethylformamide, are employed along with a hydroperoxide and an indicator; of U.S. Pat. No. 4,071,318 (use of borate esters); and of U.S. Pat. No. 4,071,321 (use of both diluents and borate esters).
Another reference of interest to these general concepts is U.S. Pat. No. 3,236,850, directed toward stabilizing organic hydroperoxides used as catalysts and oxidizing agents. This reference discloses the use of primary, secondary or tertiary amine salts with organic peroxides, and does not address the stability problems of solid phase reagent test strip devices.
A study of thermal decomposition reactions of alkyl hydroperoxides is described in J. R. Thomas, J. Am. Chem. Soc., 101, pp. 246-248 (1955). The decomposition rates of four different hydroperoxides in a hydrocarbon solvent, as a function of temperature, were measured. The inclusion of phenyl-1-naphthylamine in the solution mixture was observed to produce a decrease in the rate of hydroperoxide disappearance. In addition, J. R. Thomas and O. L. Harle, J. Phs. Chem., 63, pp. 1027-1032 (1959), discuss studies of the influence of solvents on the rate of decomposition of tetralin hydroperoxide. Phenyl-L-naphthylamine is disclosed as being used in these studies as a radical trap to inhibit radical chain reactions in the decomposition of the hydroperoxide.
These foregoing articles do not directly address stability problems of solid phase reagent strip devices. All of the foregoing reported studies were carried out in solution, rather than in solid-phase, the latter being the state of the hydroperoxides conventionally used in strip devices. Moreover, the solvents typically used in strip devices, e.g., dimethylformamide and acetone, are substantially different from those described in this literature: medicinal white oil, octane, decane, cyclohexane and decalin. Also, reagent strips typically contain reagents which potentially can deleteriously interact, not only with the hydroperoxide, but also with other strip ingredients.
Accordingly, despite the inherent analytical advantages of solid phase reagent strip devices over solution chemistry procedures, and the foregoing exemplary advances in the art of stabilizing the reactivity of such strip devices, the stability characteristics of the latter, particularly in the case of devices for the determination of peroxidatively active substances, are in need of even further improvement. Whereas the properties of current solid phase, state-of-the-art compositions and devices for determining peroxidatively active substances are greatly enhanced over those of wet chemical methods, and over those of methods including no stability-enhancement techniques, it would nonetheless be greatly advantageous if even more stability during prolonged storage could be afforded, eliminating "false negative" results while retaining adequate sensitivity to peroxidatively active analytes following such storage. Preferably, this should be accomplished without the need for isolation of reagents by encapsulation or similar relatively complex and expensive treatments of such compositions and devices. For example, it would be advantageous to provide suitable stabilizing agents which are readily commercially available, economical to use, and which would afford adequate sensitivity for otherwise conventional reagent/indicator systems used in solid phase test compositions and devices, as well as rendering the compositions and devices substantially more stable during long-term storage.
It is presently postulated that the frequently-observed losses of reactivity, leading to lack of storage stability or "shelf-life" of conventional solid phase reagent compositions and strip devices for determining peroxidatively active substances, may be primarily attributable to loss and/or chemical degradation of the organic hydroperoxide used in the reagent strip. Such loss or degradation may occur, for example, from decomposition or volatilization of the hydroperoxide, or chemical interaction with other strip constituents. However, it is now believed that degradation due to deleterious interaction may account for a substantial percentage of reactivity losses, although the mechanism causing such interaction is at the present unknown.